Unbalanced predatory communities and a lack of microbial degraders characterize the microbiota of a highly sewage-polluted Eastern-Mediterranean stream

Abstract Wastewater pollution of water resources takes a heavy toll on humans and on the environment. In highly polluted water bodies, self-purification is impaired, as the capacity of the riverine microbes to regenerate the ecosystem is overwhelmed. To date, information on the composition, dynamics and functions of the microbial communities in highly sewage-impacted rivers is limited, in particular in arid and semi-arid environments. In this year-long study of the highly sewage-impacted Al-Nar/Kidron stream in the Barr al-Khalil/Judean Desert east of Jerusalem, we show, using 16S and 18S rRNA gene-based community analysis and targeted qPCR, that both the bacterial and micro-eukaryotic communities, while abundant, exhibited low stability and diversity. Hydrolyzers of organics compounds, as well as nitrogen and phosphorus recyclers were lacking, pointing at reduced potential for regeneration. Furthermore, facultative bacterial predators were almost absent, and the obligate predators Bdellovibrio and like organisms were found at very low abundance. Finally, the micro-eukaryotic predatory community differed from those of other freshwater environments. The lack of essential biochemical functions may explain the stream's inability to self-purify, while the very low levels of bacterial predators and the disturbed assemblages of micro-eukaryote predators present in Al-Nar/Kidron may contribute to community instability and disfunction.


Introduction
In many parts of the w orld, sew age flo ws to nearb y rivers without tr eatment, tr ansforming an essential life-sustaining resource into a cause of harm to health and environment.Under high inputs of se wa ge containing high loads of pathogens, high concentrations of organic matter, chemicals and mineral nutrients, water quality deteriorates as the capacity of the riverine microbes to regenerate the ecosystem is overwhelmed (Pascual-Benito et al. 2020 , Red d y andDube y 2021 ).While recent studies show that se wa ge dr asticall y alters the aquatic microbiome's composition and dynamics (Korajkic et al. 2015, Zhang et al. 2015 ), information on the composition, dynamics and functions of microbial communities in highl y se wa ge-impacted riv ers, whic h ar e common in many parts of the world, is restricted.In that sense, w astew ater treatment plants (WWTPs) provide a valuable comparison with highl y se wa ge-polluted str eams as their r aw material is similar.Yet in WWTPs, se wa ge is efficiently purified to reclaimed water by the plant's microbial communities, which are under ecologi-cal selection by the constructed environment and the operational conditions (Wang et al. 2012, Cohen et al. 2019 ).Depending on the quality of the released reclaimed water into the environment, micr obial comm unities ar e mostl y locall y disturbed (Kor ajkic et al. 2015, Pascual-Benito et al. 2020 ).Numerous studies have shown that WWTPs are enriched in populations performing tasks such as nitrification, organic material breakdown, phosphate accumulation and m uc h else (Ju andZhang 2015 , Cohen et al. 2019 , among others).Also, fecal and pathogenic micr oor ganisms decay, for example, through competition, predation and exposure to unfit conditions (Korajkic et al. 2019 ).
Predation is thought to play an important role in shaping the dynamics and composition of bacterial communities in different envir onments (Chauhan 2009;Sha pir o et al. 2009, Johnke et al. 2014, Hungate et al. 2021 ), but lar ge-scale and time-series anal yses of microbial predators are rare and almost exclusively focus on micro-eukaryotes (Pauli et al. 2001, Madoni 2011, Zahedi et al. 2019 ).Latel y, the gener alization of high-thr oughput 16S rRNA gene community analysis has uncovered the ubiquitous presence and r esponsiv eness of bacterial pr edators suc h as the bacterial predators Bdellovibrio and like organisms (BALOs) (Chauhan 2009, Feng et al. 2017, Cohen et al. 2021, Hungate et al. 2021 ).They appear to form v ery div erse, common comm unities able to pr ey on a v ery lar ge v ariety of bacteria (Kandel et al. 2014, Feng et al. 2017, Cohen et al. 2021 ).
So far, very few studies have examined community composition and dynamics of raw sewage-highly impacted rivers .T he aim of this study was to fill that knowledge gap and to address the hypothesis that the self-purification potential of such streams is undermined because biochemical and ecological functions provided by the bacterial and by the micro-eukaryote communities ar e lac king.In order to ac hie v e this goal, the highl y se wa gecontaminated Al-Nar/Kidron (ANK) intermittent stream that originates in Jerusalem and flows to the Dead Sea ( Fig. S1 ) was sampled for 1 year.We used rRNA-gene high-throughput sequencing to analyze the microbial community at large, including Bacteria, and more specifically BALO predators, and the micro-eukaryotes, while monitoring the chemical and physical environmental parameters .T he results of these analyses were contextualized versus published data from other rivers and from a WWTP located in Al-Bireh (Cohen et al. 2019 ), distant by ∼25 km, and fed inputs similar to those entering the ANK stream.

Sampling
The ANK river is a seasonal, winter stream with its head in Wadi J oz in J erusalem (750 m).It flows eastward on a 34 km-long course to r eac h the shor es of the Dead Sea at Avnat (-415 m; refer to Figure S1 for more details, see Supplementary materials and methods ).Surface river (0-15 cm) water was collected in 1-L sterile jars in triplicate .T he samples were k e pt on ice until delivery to the laboratory within a few hours, for chemical and physical analysis at Bethlehem University.The rest were refrigerated and tr ansferr ed on ice to the Hebr e w Univ ersity within 24 h, for DNA extraction and further processing.Sampling was performed once during the first week of the month for 1 year from March 2013 to February 2014 and, additionally, for four consecutive weeks in August 2013 and in February 2014, at sites along a str etc h of ∼12 km, in and near Al-Ubeidiya, depending upon accessibility Figure S1B ).

DN A extr action
Two ml of water of eac h r eplicate fr om eac h sampling time were centrifuged at 10 000 g for 10 min at 4 • C, washed with sterile double distilled water and centrifuged again.The pellet was suspended at 1.2 ml of double distilled water (DDW) and then processed using a Po w erSoil TM DN A Extraction Kit (MoBio Laboratories, USA), according to the manufacturer's instructions.DNA concentration and purity were measured using Nanodrop TM 2000 (T hermo Scientific , Wilmington, DE, USA) and was in the range of 20-100 ng.μl −1 for all samples.

Bacterial quantification by real-time qPCR
qPCR was performed as in Cohen et al. ( 2021 ).Briefly, standards wer e pr epar ed by inserting a 1467-bp fr a gment of the Bdellovibrio bacteriovorus HD100 and Bacteriovorax stolpii UKi2 strain 16S rRNA gene amplified with primers 27F and 1492R, r espectiv el y, into a PGEM-T easy plasmid vector system (Promega, WI, USA).Ten-times serial dilutions from 10 3 to 10 10 plasmid copies per reaction were used to construct standard qPCR curves and plasmid copy numbers were calculated.For total bacteria, the primer pair 1048F-1175R was used to quantify the 16S rRNA gene copy n umber.For ad ditional details, see Supplementary materials and methods .

Results and discussion
Av er a ges of 46 K of and 16.2 K reads per sample were obtained for Bacteria 16S rRNA and Eukarya 18S rRNA gene-targeted sequencing, r espectiv el y, enabling almost complete cov er a ge of the comm unities ( Figur e S2A-D ).The number of Bd and Bx amplicons obtained was lower (7924 and 2920, r espectiv el y), yet, for both BALOs, high cov er a ge based on Good's cov er a ge was ac hie v ed ( Figure S2B-C ).
Total bacteria in the ANK str eam, as r ec k oned by 16S rRNA copy numbers, w as high ( ∼2.10 9 cop y/ml) (Fig. 1 A), on a par with those measured in the liquor (the effluent-like fraction) of WWTP secondary treatment reactors (Cohen et al. 2019 ).Diversity of the Bacteria and of the micro-eukaryotes was lower than in other rivers, riv er systems, micr ocosms impacted by WWTP-effluent or by urban water (Table 1 and Table S1 ) (Korajkic et al. 2015, Li et al. 2018, Wang et al. 2018, Cohen et al. 2019, Nakatsu et al. 2019, Pascual-Benito et al. 2020, Xu et al. 2020, Abdullah Al et al. 2021, Muhammad et al. 2021, Shang et al. 2021, Ting et al. 2021 ).More specificall y, the α-div ersity estimates of the ANK str eam's Bacteria and of the micro-eukaryote communities were significantly differ ent, but their v alues wer e m uc h closer than those observ ed in the nearby Al-Bireh (AB) WWTP (Table 1 ) (Cohen et al. 2019 ).Furthermor e, Br ay-Curtis distances of both microbial communities were significantly lo w er than those calculated from the AB WWTP data (Cohen et al. 2019 ) (Bacteria: ANK, 0.372; AB flocs, AB liquor: 0.548, 0.777; micro-eukaryotes: ANK, 0.275; AB flocs, AB liquor: 0.707, 0818, P < 0.0001).This can be explained by the differ ent bacterial comm unity structur es between ANK and AB, and by the strong seasonal variations observed in the ANK river data (MRPP-A = 0.223, P < 0.001, Figure S3A, B ). T hus , the ANK river's micr obial comm unities exhibited r educed div ersity and r educed stability compared with other fresh water bodies in which sewage pollution was less se v er e.More than 90% of the operational taxonomic units (OTUs) detected in the ANK samples were affiliated to the Proteobacteria (Fig. 1 B).γ -and ε-proteobacteria largely dominated, comprising ∼90% of the sequences .T hey wer e mainl y composed of members of the families Moxarellaceae ( γ -proteobacteria, mostly Acinetobacter ) and Campylobacteriaceae ( ε-proteobacteria, almost exclusiv el y Arcobacter ).Moxar ellaceae and Campylobacteriaceae r eac hed maxima in winter (r elativ e abundance RA = 0.58), and in summer (RA = ∼0.87),and their lo w est le v els in summer (RA = ∼0.03RA, significant difference with the former's summer maxim um, Wilcoxon r ank-sum, P = 0.011) and in winter (RA = 0.23; no significant difference with the latter's summer maximum, Wilcoxon rank-sum, P = 0.5238), r espectiv el y (Fig. 1 C).This pattern was in line with the significant Pearson correlations between bacterial comm unity structur e anal ysis and temper atur e ( Table S2 ).Other, w eaker sour ces of variation (( | r | ) > 0.45) included salinity and nitrate ( Table S2 ).The high TSS and BOD values pointed to the high le v els of organic compounds present in the water.They were higher than those measured in the liquor fraction of WWTPs (Cohen et al. 2021 ), and above the limits of ∼30 mg.l −1 set for dir ect envir onmental waste water disc har ge (UK Envir onment Agency 2019 ) ( Table S3 ).The γ -and ε-pr oteobacteriaar e numerically important classes in WWTPs, as are the βproteobacteria (Ju and Zhang 2015 , Saunders et al. 2016, Cohen et al. 2019 ).The lat-ter are also common in the water column of rivers (Zeglin 2015 ), but in the ANK stream, they were detected at low levels at all times (0.08 ≥ RA ≥ 0.026).Although Bacteroidetes diversity was consequent (227 OTUs), these bacteria were present at low levels (0.06% ± 0.001%) (Fig. 1 C, Table 2 ).Bacter oidetes ar e found at high abundances in WWTPs and in the water column of streams, e v en in pollution/se wa ge-impacted ones (Zeglin 2015 , Jordaan andBezuidenhout 2016 ).The large number of peptidases, glycoside hydrolases and glycosyl transferases their genomes encode for (Fernández-Gomez et al. 2013 ) allow them to grow attached to organic matter particles and hydr ol yze them, as well as to degrade dissolv ed or ganic material.Her e, Acinetobacter spp.wer e pr esent at a high RA.They can degrade aromatic compounds and hydrocarbons, and many strains are lipolytic (Jung et al. 2015 ); Aeromonas spp.wer e conspicuousl y pr esent ( > 1.8% RA) and can degr ade chitin (Seshadri et al. 2006 ).Together, this suggests that some organic compounds can be degraded in ANK water, but these functions do not replace those performed by the missing species (Table 2 ).Other taxa playing important roles in water purification, suc h as nitrification, denitrification, hydr ol ysis and phosphorus accum ulation, wer e also found at low to very low le v els in the ANK samples (Table 2 ) compared with WWTPs (Ju and Zhang 2015, Cohen et al. 2019, Zhang et al. 2019 ).More specifically, the nitrifiers Nitrosomonas and Nitrospira, and Nitrospina and Nitrotoga , were hardly detected or were not found at all, respectively (Table 2 ).This suggests a low potential for nitrification, which could explain the r elativ el y high ammonium le v els ( Table S3 ).In polluted rivers, ammonium concentration was found to have a stronger influence on the composition of microbial communities than did nitrate, the le v els of whic h wer e also high in the ANK stream ( Table S3 ) (Shang et al. 2021 ), maybe because denitrifiers were one or more orders of magnitude lo w er than in WWTPs (Table 2 ; Cohen et al. 2019 ).

ANK
T his , despite the high density of Arcobacter and Acinetobacter , species that include efficient denitrifying strains that can be gr eatl y enric hed for in nutrient r emov al or sludge-based denitrification cultures (Pishgar et al. 2019, Xia et al. 2020 ), suggests that these were not selected for or not active in the stream.Phosphate accum ulators wer e r ar e, as Tetr asphaer a were absent and Accumulibacter was very scarce (Table 2 ).In that sense, the high concentrations of Acinetobacter may (partly) fill that role and reduce phosphorus to le v els (6.3 ppm) in the r ange of WWTP effluents (5-20 ppm) (Cloete et al. 1985, Yeoman et al. 1988 ).Three common clades of complex organic compound degraders in WWTPs were almost completely absent in ANK, despite the high organic loads: Myxococcales (0.2%), which include n umerous facultati ve predators (Shimkets 1990 ); floc-forming Chloroflexi (0.03%), which feed on lysed bacterial cell debris (Speirs et al. 2019 ); and Planctomycetes (0.02%), which mainly associate with particles, surfaces and hosts (Wiegand et al. 2018(Wiegand et al. , 2020 ) ). Human fecal bacteria, on the other hand, did not appear to be more represented in the ANK water samples than in the AB and other WWTP samples (Cohen et al. 2019 ).Yet, the v ery high le v els of the opportunistic pathogens Acinetobacter and Aeromonas should certainly be noted.
The identified taxa in the micro-eukaryote community analysis accounted for 31.7% of the total O TUs .T he Opisthokonta (animalia, fungi) accounted for 43.6% of identified sequences, follo w ed b y the Str amenopiles, Alv eolates and Rhizaria (SAR) Super gr oup (11.4%), and the Archaeplastida (algae , plants , 5%) (Fig. 2 A).The most dominant ( > 1%) OTUs are listed in Table 3 , and accounted for 71.3% of total RA, except for the low e v enness value (Table 1 ).Most were unidentified, and the identified taxa wer e mostl y not pr edatory.Y et, among all sequences, numerous potential bacterial predators were identified ( Table S4 ; in to-tal, 238 O TUs).T hey w ere dominated b y SAR Ciliophora, Excata and Amoebozoa.Among those, dominant OTUs included three P ar abodo (Excav ata) and two Hymenostomatia (SAR) that gr eatl y v aried thr oughout the year, peaking at 16.7%, 13% and 4.45% in September, and at 25% and 19.5% in December, r espectiv el y (Fig. 2 B-H).Ar ac hnids, r otifers and nematodes, whic h can also consume bacteria and protists, accounted for 4% of the sequences.Remarkably, most OTUs exhibited maximal RA from the end of Se ptember to February, exce pt for Rhizaria and some non-fungi Opiskhonta (mostly Metazoa) that fluctuated during the whole year (Fig. 2 B-H).
These analyses of bacterial and micro-eukaryote communities and of ANK river water support the notion that heavy se wa ge pollution along with extreme climatic conditions (Zituni et al. 2021 ) resulted in specific assemblages unable to provide water-purifying functions such as clarification, biochemical oxygen demand and turbidity reduction by organic matter (OM) degradation and nitrification, whic h ar e known to depend upon balanced pr edatory activities (Madoni 2011 ).In turn, the restricted potential for degradation suggests that the ANK river's microbes mostly tolerate but do not degrade the contaminants (Chakraborty and Bhadury 2015 ).
In order to further address the potential role of bacterial predators in this se v er el y polluted ecosystem, a BALO-tar geted 16S rRNA gene community analysis was performed.It revealed that Bbellovibrionaceae (Bd) and Bacteriovoracaceae (Bx) diversities w ere lo w er than those observ ed in WWTPs, r espectiv el y (Cohen et al. 2021 ) (Table 4 ;t-test, P < 0.01, two-tailed).The BALO predators wer e pr esent at v ery low r elativ e abundances in the 16S rRNA gene reads of the general Bacteria, averaging 0.004% ± 0.00085% for the Bd and 0.009% ± 0.001% for the Bx.These low le v els wer e confirmed by qPCR targeting the Bd community (Fig. 1 ).T hus , while the diversity of the BALO community (Bd + Bx) was lowerbut not extr emel y differ ent fr om that observ ed in r ele v ant habitats (WWTPs and freshwater) (Paix et al. 2019, Ezzedine et al. 2020, Cohen et al. 2021 )-its abundance was gr eatl y r educed.The abundance of Gr am-positiv e taxa (non-prey) or a negative effect of the high organic matter content in the river as possible causes for the low abundance of BALOs cannot be invoked as Gr am-positiv e cells constituted only ∼15% of the bacterial community (Firmicutes and Actinobacteria mostl y).Mor eov er, BALOs ar e abundant in WWTP sludge in which the content of organic matter is very high (Feng et al. 2017, Cohen et al. 2021 ).In effect, BALOs appear to be more abundant in or ganic matter-ric h envir onments, pr obabl y because these sustain large bacterial communities that may be preyed upon, such as in WWT schemes or in soils with a high organic matter content (Jurkevitch 2020 , Petters et al. 2021 ).A possible clue to the low BALO abundance in the ANK river may be the high ammonium concentrations ( Tables S3 and S5 ), which w ere found b y Paix et al. ( 2019 ) to negativ el y corr elate with Bd populations in peri-alpine lakes.Other potential causes could be pr edation-inhibiting substances suc h as or ganophosphates, and Table 2. Heatmaps of minimum, maximum and averaged relative abundance (RA), standard deviation and occurrence in samples (%) of bacterial taxa found in the Al-Nar/Kidron (ANK) stream, and known to perform specific ecological functions according to Ju and Zhang ( 2015 ).RA values of heatmaps and occurrence (%) values are presented in the last row and right most column, respectively.Species in or ange-color ed cells are missing or found at very low levels; in white cells, they are at levels comparable with WWTPs ( ± 1 order of magnitude) (Cohen et al. 2019 ); in the light green cells, these are species at higher levels than in the ANK stream.herbicides used in a gricultur e, household and industrial detergents and surfactants, and fecal matter-associated compounds like indole, as well as antibiotic residues (Mitchell et al. 2020 ), all of which may be present in the ANK river water.
Temper atur e exerted a strong effect upon ANK's Bdellovibrionaceae populations, and, to a lesser extent, nitrate ( Table S5 ).Among the dominant Bd OTUs ( > 0.1% in > 90% of the samples), Bd1 was found year-long at high abundance with a population peak during the summer months; by contrast, populations of Bd16 and Bd22 were enhanced during the colder period.OTU Bd30 was mostly stable.Y et, summer -promoted or winter -promoted OTUs could exhibit large fluctuations (Fig. 3 A).The temper atur e dependence of BALO communities has been noted numerous times (Staples and Fry 1973, Williams 1988, Paix et al. 2019, Yan et al. 2022 ), but its impact on the predator vs. indirect effects due to environmental c hanges (e.g.pr ey comm unity structur e, water composition) needs to be e v aluated.Other dominant Bd OTUs shifted on a shorter time-frame (e.g.Bd1, Bd22, Bd24).A possible cause may be r egulation by pr ey av ailability (Chen et al. 2011 ) or by Kill the Winner dynamics (Sha pir o et al. 2009, Winter et al. 2010, Cohen et al. 2021 ).It can be noted that, r espectiv el y, Bd1 and Bd16 were found in all, and almost all core BALO communities in the floc and liquor fr actions of thr ee WWTPs (data fr om Cohen et al. 2021 ), suggesting that these populations are not geographically restricted, which may be due to an ability to sustain rather different environmen-tal conditions.Phylogenetic analysis sho w ed that very few of the Bd OTUs wer e r elated to the "classical" Bd strain B. bacteriovorus H-D100, which was isolated from soil (Stolp and Starr 1963 ).One OTU was closel y r elated to the soil isolate B. reynosensis (Ajao et al. 2023 ), and three other Bd OTUs were in sister clades to known isolated r epr esentativ es ( Fig. S4 ).By contr ast, onl y two Bx OTUs dominated, showing inverted patterns of abundance between summer and winter (Fig. 3 B).Yet, we car efull y suggest that other par ameters, not included in the ones measured in the current study, may pr e v ent the establishment of Bx populations.

Conclusion
In an intermittent Mediterranean stream where WWTP effluent (e.g. with lar gel y r educed or ganic loads compar ed with those found in the ANK river) could constitute up to 100% of the streamflow during the summer period, the riv er's micr obial comm unities wer e r a pidl y r estor ed within 1 km (P ascual-Benito et al. 2020 ).By comparison, the sampled str etc h of the ANK river that remains se v er el y polluted extends for more than 10 km and is situated more than 15 km a wa y from the main source of sewage.
Our data, showing that the ANK river's microbiota are deficient in OM degraders and in nitrifiers, point at a largely reduced potential for water self-purification.In a recent study, Shang et al. ( 2021 ), using alternative stable state theory, found that, in Table 3. Relative abundance (RA; annual av er a ge and standard error, in %) of the most abundant micro-eukaryote single OTUs above 1% RA.When a vailable , taxonomic affiliation is at the family/genus level, along with a general description of feeding habits.addition to higher div ersity, co-occurr ence patterns of microbial communities in favorable (less impacted) river environments included Myxococcales and NItr ospir ales as k e y taxa, with Rhizobiales also forming large hubs.Our data show large fluctuations in comm unity structur e betw een sampling times, lo w e v enness and low div ersity, whic h indicate low ov er all stability; they also r e v eal a scarceness of BALO and Myxococcales predators, an absence or very low presence of Rhizobiales, Bacterioidetes and Planctomycetes, all of which are not only important degraders of organic compounds, but also large components in BALO co-occurrence networks in WWTPs and in freshwater (Feng et al. 2017, Ezzedine et al. 2020, Cohen et al. 2021 ).We thus further propose that the dearth of both facultative and obligate bacterial predators, along with the specific composition of the predatory micro-eukaryote comm unity pr esent in the stream, hamper the establishment of efficient trophic networks that help stabilize communities (Welsh et al. 2015, Cohen et al. 2021, Hungate et al. 2021, Lee et al. 2021, Petters et al. 2021 ) and contribute to increased community diversity (Winter et al. 2010 ).We propose that future studies on water self-purification of highl y se wa ge-impacted str eams may benefit from accentuated research on the role of micro-predators in reestablishing functional, water-purifying assemblages (top-down) vs. water compositional and environmental parameters and their effects on microbial degraders and recyclers (bottom-up).As for the ANK riv er, fortunatel y a r estor ation pr oject has been initiated, and the river's water and riverine habitats are expected, if not to r ecov er, then at least to significantly improve in future years (HaGihon 2023 ).

Figure 1 .
Figure 1.(A) Quantitative PCR of the 16S rRNA gene copy number for Bacteria and Bdellovibrio during a year-long sampling effort at the Al Nar/Kidron (ANK) stream.(B) Distribution of taxonomically classified 16S rRNA gene-based OTUs at the family level.(C) Yearly dynamics of the dominant bacterial clades at the ANK stream; the Greek letters refer to classes in the Proteobacteria.
Table 1.α-diversity estimates (S = Richness , E = Evenness , H = Shannon index, D' = Simpson index), and standard error, of bacterial and eukaryotic communities of the Al-Nar/Kidron (ANK) river, calculated based on OTU grouping of 16S rRNA gene and 18S rRNA gene sequences .T he Al-Bireh (AB) WWTP data (italics) ar e fr om Cohen et al. ( 2019 ) and ar e pr ovided for comparison; F, floc fraction; L, liquor fraction.

Figure 2 .
Figure 2. (A) Pie r epr esentation of the distribution of identified microeukaryote phyla.(B-H) Dynamics of all OTUs in microeukaryote phyla/classes/orders along the year-long time series in the ANK stream.In Excavata and in the SAR, Ciliophora, highly abundant OTUs at specific time points are shown as short red bars, with relative abundance (RA) in %.

Figure 3 .
Figure 3. Ann ual d ynamics of the most abundant Bdello vibrionaceae (Bd) (A) and Bacterio voracaceae (Bx) (B) O TUs throughout the year-long time series in the ANK stream.